Fluorescence technology has revolutionized cell biology and many areas of biochemistry. In certain instances, fluorescent molecules may be used to trace molecular and physiological events in living cells. Certain sensitive and quantitative fluorescence detection devices have made fluorescence measurements an ideal readout for in vitro biochemical assays. In addition some fluorescence measurement systems may be useful for determining the presence of analytes in environmental samples. Finally, because certain fluorescence detection systems are rapid and reproducible, fluorescence measurements are often critical for many high-throughput screening applications.
The feasibility of using fluorescence technology for a particular application is often limited by the availability of an appropriate fluorescent sensor. There are a number of features that are desirable in fluorescent sensors, some of which may or may not be present in any particular sensor. First, fluorescent sensors should produce a perceptible change in fluorescence upon binding a desired analyte. Second, fluorescent sensors should selectively bind a particular analyte. Third, to allow concentration changes to be monitored, fluorescent sensors should have a Kd near the median concentration of the species under investigation. Fourth, fluorescent sensors, especially when used intracellularly, should produce a signal with a high quantum yield. Fifth, the wavelengths of both the light used to excite the fluorescent molecule (excitation wavelengths) and of the emitted light (emission wavelengths) are often important. If possible, for intracellular use, a fluorescent sensor should have excitation wavelengths exceeding 340 nm to permit use with glass microscope objectives and prevent UV-induced cell damage, and possess emission wavelengths approaching 500 nm to avoid autofluorescence from native substances in the cell and allow use with typical fluorescence microscopy optical filter sets. Finally, ideal sensors should allow for passive and irreversible loading into cells.
The importance of metals in biological systems and the general difficulty in measuring metals in living cells makes metal detection a particularly desirable field for the use of fluorescence technology. As one example, zinc is a vital component in many cellular processes. Although the traditional study of the bioinorganic chemistry of Zn2+ has focused on structural and enzymatic functions in proteins, the neurobiology of Zn2+ has been gaining attention. Whereas most Zn2+ in biological systems is tightly bound in proteins and enzymes, a pool of free Zn2+ has been imaged in cells. Sub-nanomolar concentrations of Zn2+ have been detected in undifferentiated mammalian cells, and higher concentrations, approaching 300 μM, have been imaged in the mossy fiber terminals of the hippocampus. The Zn2+ ion has the ability to modulate a variety of ion channels, may play a role in neuronal death during seizures, is pertinent to neurodegenerative disorders, and may be vital to neurotransmission and long-term potentiation.
Although Zn2+ is critical to cellular processes, excess zinc ions may be toxic. The levels of Zn2+ in the brain and other parts of the body are believed to be regulated by three related Zn2+ transport proteins (ZnT-1, ZnT-2, and ZnT-3) and by metallothioneins (MTs), including MT-III and MT-IV which are expressed mainly in the brain. ZnTs and MTs are probably responsible for distributing the required Zn2+ to proteins and enzymes, and minimizing the amounts of free Zn2+ present in cells. In nerve cells, however, free Zn2+ is available for neurological functions because Zn2+ can be released from synaptic vesicles and can enter cells through voltage-dependent Ca2+ channels. Despite the abundance of research, many aspects of ionic Zn2+ in neurobiology remain unclear due to the limited detection methods currently available.
Because metal ion levels may be critical to normal cellular function, a number of diseases may result from, or may be caused by, errors in metabolism of a particular metal ion in the affected individual. For example, abnormal zinc metabolism has been found in some Alzheimer's patients, and low levels of zinc are associated with various behavioral disorders. Diagnosis of errors in such metal ion metabolism may be facilitated by the subject invention.